Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses

Summary Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAV) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for a N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.


Introduction
Influenza virus is a highly mutable pathogen that presents an ongoing diversity challenge to the immune system.A core issue is that hemagglutinin (HA), the major viral spike protein and seasonal vaccine immunogen, preferentially elicits antibodies against its hypervariable regions, limiting coverage and compromising pandemic preparedness [1][2][3][4] .Of the four influenza virus types (A, B, C, D), influenza A viruses (IAV) are responsible for a majority of infections in humans and are the source of influenza pandemics [5][6][7] .IAVs are phylogenetically divided into group 1 and group 2 viruses, and intragroup diversity is further subdivided into subtypes categorized by 18 distinct IAV HAs 8,9 .Broadly neutralizing antibodies (bnAbs) covering different levels of IAV diversity can be generated by targeting invariant features of HA, however these responses are immune-recessive/subdominant and will require rationally designed immune-focusing concepts to elicit higher titers 1,2,[10][11][12][13] .
The majority of HA sequence diversity resides in its globular head domain, and 'universal' vaccine concepts currently under clinical evaluation include antibody-focusing to the relatively conserved stem or stalk domain of this protein [14][15][16] .These vaccines involve structure-based presentation of stem-only nanoparticles or sequential immunization with chimeric/head domain varying HAs to boost serum antibodies against conserved stem epitopes.Monoclonal human bnAbs can engage these sites to provide broad levels of coverage: heterosubtypic and the more exceptional cases of cross-group protection 2,[17][18][19][20][21][22][23][24][25] .Within preclinical vaccine models, protective activity likely involves infection-blocking and Fc-mediated effector functions that are maximized when the bnAb sites are engaged [26][27][28][29] .
A feature of vaccine expanded stem responses in preclinical models is that coverage remains heterosubtypic and is not cross-group 28,[30][31][32][33] .Differences between the glycosylation positions on the group 1 vs group 2 stem also impose steric constraints that hinder intergroup-reactivity to bnAb supersites 32,34,35 .There is no single stem immunogen that has been shown capable of eliciting intergroup IAV bnAbs.Rather, intergroup coverage has required co-formulation of group 1 + group 2 stem immunogens 36 .
In this study, we sought to elicit intergroup protective antibodies using a single vaccine immunogen by expanding germline BCR precursors that give rise to these bnAbs in humans.Functionally convergent human intergroup bnAbs are generated through public/ shared antibody classes marked by usage of specific V H genes, D H genes and/or CDRH3 signatures 20,22,24,25 and they have the potential to be triggered and expanded in vivo by different stem-focusing vaccine modalities 37,38 .We applied our one-step CRISPR/Cas9induced homology-directed repair (HDR) platform 39,40 to produce a humanized mouse vaccine model containing physiologically relevant recombination frequency of germline BCRs giving rise to intergroup bnAbs of the VH1-18 QxxV class 24,37 .We then immunized this mouse with recombinant nanoparticle displays of the group 2 HA stem 37 , which we found harbor natural binding affinity to VH1-18 QxxV bnAb precursors.The particles induced protection against group-unmatched IAV, and this was dependent on the bnAb precursors within our humanized mice.We found that after a single immunization, bnAb precursors were recruited to B cell germinal centers (GC) and underwent somatic hypermutation (SHM).Within 28 days post vaccination, potent and cross-protective bnAbs were elicited, and with only a fraction of mutations seen in the mature antibody from humans.Notably the immunogens enriched for N55T, a CDRH2 mutation present in all human VH1-18 QxxV bnAbs.We show that this mutation alone enables cross-group protection by providing a flexible fulcrum to pivot the antibody and accommodate the conserved glycosylation differences between the group 1 and 2 HA stems.Our findings thus reveal a simple molecular switch that can be triggered with minimal antigenic input to elicit exceptionally broad humoral immunity against IAV.

Physiological B cell repertoire frequency of VH1-18 QxxV bnAb precursors enables vaccine-protection from group-unmatched IAV
H3ssF elicits heterosubtypic immunity within group 2 IAV, but this does not extend to group 1 IAV 37 ; coformulation of group 1 and group 2 immunogens is needed for cross group protection 36 .To initially define whether the presence of VH1-18 QxxV bnAb precursors (at human-like recombination frequency, see also Figure 2E-F) could enable cross group protection, we sequentially (2x) immunized animals containing (or not containing) 09-1B12-UCA B cells (at human precursor frequency) with H3ssF (+ Sigma adjuvant) and then lethally challenged with subtype-matched H3N2 or group-unmatched H1N1 influenza viruses (Figure 2H).We found that H3ssF protected against subtype-matched H3N2 virus in the absence of UCA cells, but required their presence for protection against the groupunmatched H1N1 virus.Hence, the inclusion of VH1-18 QxxV bnAb precursors into a diverse host B cell repertoire at human-like recombination frequency enabled the elicitation of cross-group protective immunity, all using a single recombinant vaccine immunogen.

Individual stem nanoparticles selectively expand and mature the bnAb precursors within B cell germinal centers in the humanized mice
To investigate the B cell pathways underscoring cross-group protection, we tracked CD45.2 VH1-18 QxxV UCA lineage expansion after a single immunization.In these experiments, we deployed H3ssF and H7ssF 37 as higher and lower affinity germline stimulating immunogens (Figure 3A); monomeric UCA BCR affinities for the H3 and H7 stems from these antigens is separated by a logfold difference (1.24e-07 vs 2.68e-06 M) (see also Figure 1D, Table S3).H3ssF and H7ssF (+Sigma adjuvant) triggered comparable recruitment of CD45.2 B cells to germinal centers (GCs) within the CD45.1 hosts after one immunization step (Figure 3B-D, Figure S5A,B).Naked ferritin particles were also injected with Sigma Adjuvant (Figure 3A) and while this elevated GC reactions in the CD45.1 host (Figure 3B,C), it was not accompanied by recruitment of CD45.2 UCA B cells (Figure 3D).Thus, expansion of the CD45.2UCA B cells was dependent on the stem-antigens.We further confirmed that GC-recruited CD45.2 B cells were targeting the central stem epitope (H3ssF + /H7ssF + /H3ssF-KO − /H7ssF-KO − , epitope KO = insertion of N-linked glycan at 45 HA2 ) (Figure 3E, S5A,B).If we exchanged our nanoparticle flow cytometry probes for H7 and H3 trimers, we recapitulated our key findings: expansion of H3 + /H7 + cross-reactive CD45.2 B cells into GCs at 8, 15 and 28 days after immunizing with H3ssF or H7ssF, and failure to trigger this response if ferritin alone is deployed as the immunogen (Figure S5C-G).
Single cell BCR sequencing of the CD45.2 + /CD45.1 − /H3ssF + /H7ssF + /H3ssF-KO − / H7ssF-KO − GC B cells revealed diversification through SHM in response to both H3ssF and H7ssF (Figure 3F-K; Table S4).SHM was concentrated in the CDR1-3 regions of the HC and LC (Figure 3H-K), and mutations also accumulated in FW3 of the HC and LC (Figure 3H-K).Notably, the mutations N55T (CDRH2) and S32R (CDRL1) enriched in the vaccineexpanded BCRs (Figure 3H-K) are also present in the mature 09-1B12 and 16.g.07(Figure 1B,C).In the mature antibodies, contact to the group 1 stem is greatly strengthened by S32R in CDRL1 (Figure 1).The importance of N55T, a hallmark of the VH1-18 QxxV bnAb class in humans 24 , is detailed in the following sections.Collectively, these data indicate that our immunogens: (A) selectively trigger and expand the VH1-18 QxxV bnAb lineage from a diverse B cell repertoire bearing bnAb precursors at human-like recombination frequency; and (B) guide SHM and affinity selection to enrich for some hallmark mutations seen for this bnAb class in humans.
The stem nanoparticles elicit cross-group protective bnAbs with minimal SHM after a single immunization.
We expressed two example mAbs (O1 and O2) that were expanded by H3ssF or H7ssF, respectively (Figure 4A, Table S4).These antibodies harbored some of the amino acid mutations present in mature 09-1B12, however they also showed far lower SHM (Figure 4A).Despite this, these vaccine elicited antibodies showed 09-1B12-equivalent activities: (1) comparable neutralization activity across group 2 IAV, along with more limited neutralization of group 1 IAV, as expected for the VH1-18 QxxV bnAb class 24,46 (Figure 4B); (2) comparable protection against H3N2 viral challenge following passive transfer to C57Bl/6 WT mice (Figure 4C); and (3) comparable protection against H1N1 viral challenge following passive transfer to C57Bl/6 WT mice (Figure 4D).These results indicate that cross-group protective antibodies were elicited after a single immunization, and achieved fully functional somatic activity with minimal SHM.

Enrichment of N55T within the CDRH2 of germinal center BCRs and serum antibodies reveals a simple molecular switch conferring group 1 + group 2 IAV protection
The H3 + /H7 + cross-reactive 09-1B12-UCA BCRs expanded within GCs were enriched for the mutation N55T within the CDRH2 loop (Figure 3H,J, Table S4).Skewed use of this mutation was seen at 28 days post-immunization with H3ssF or H7ssF, but it also enriched earlier at 15 days when the higher affinity H3ssF was used as the immunogen (Figure 5A,B, Table S4).This enrichment was also observed in the serum antibodies elicited following sequential immunization with either H3ssF or H7ssF (day 0 prime + day 42 boost), as evaluated by cryoEMPEM 47,48 at 15 and 28 days post-boost (Figure 5C-H, S1, Table S1).At these timepoints, the serum antibodies had specificity to the central stem epitope, as judged by differential reactivity to H3ssF/H7ssF ± stem epitope KO (N-linked glycan at 45 HA2 ) (Figure 5C,F).For cryoEMPEM, Fabs purified from the immune sera were complexed with HA trimers matched to H3ssF or H7ssF (A/Perth/16/2009 and A/ Shanghai/02/2013, respectively), confirming antibody targeting to the central stem (Figure 5D,G, I).The significant differences of the local map resolutions at CDRH2 loop as well as the volumes around residue 55 between H3 and H7 complexes suggest H7 preferably binds to Fabs that harboring T55 but not N55, which is consistent with the binding affinity data (KDs of H7 binding to 09-1B12-UCA vs 09-1B12-UCA + N55T, Table S3).Notably, when the same H3ssF or H7ssF immunization regimens were applied to WT C57Bl/6 mice (not bearing 09-1B12-UCA cells), we failed to resolve Fab density for the full-length H3 or H7 trimer, further underscoring importance of B cell repertoire in eliciting antibodies against the central stem epitope (Figure 5E,H).When the bnAb precursors are in place, our results highlight N55T as a vaccine-selected mutation, both in the GC and serum antibody response.
Given that N55T is a marker of the VH1-18 QxxV bnAb class in humans 24 , we initially assessed the consequences of this mutation by performing molecular dynamics (MD) simulations of the 09-1B12-UCA in complex with H3 or H7 trimers (Figure 5J).In both scenarios T55 increased the interaction time and interaction areas of the QxxV motif, and also increased LC interactions with HA (Figure 5J).Accordingly, we biochemically evaluated the contribution of N55T to recognition of both group 2 and group 1 HAs for UCAs inferred from six different human VH1-18 QxxV class bnAbs (reversion of sHsL to gHgL) (Figure 6A).We found that for all antibodies, N55T strengthened germline-encoded antibody affinity to group 2 HA and also enabled binding to group 1 HAs, which was otherwise undetectable with the UCA (Figure 6A, Table S3).Alone, N55T also provided broad neutralizing activity against group 2 IAV (Figure 6B), a hallmark activity of mature VH1-18 QxxV bnAbs 24,46 .To define whether this single mutation alone enabled broad cross-group protection, we performed passive antibody transfer of 09-1B12-UCA mAb ± N55T into C57Bl/6 WT mice and then challenged with lethal doses of H3N2 or H1N1 viruses (Figure 6C,D).We observed a similar result after challenging from both viruses: the UCA form of the antibody failed to protect, however addition of N55T provided cross-group protection for >50% of recipient animals, approaching the activity of the mature 09-1B12 (Figure 6C,D).Hence, this single vaccine-selected mutation could alone enable cross-group protection from IAV.
N55T provides a 'fulcrum release' to accommodate conserved group 1 N-glycans, enabling dual recognition of group 1 and group 2 stems.
By overlaying the complexes of H3 + UCA or 09-1B12, a shift in Fab binding angles was observed, driven by the movements of HC N55T and LC S66R mutations (Figure 7A, S1).Notably the CDRH3 loop (QxxV) is at the fulcrum of this rotation (Figure 7A).This flexibility around the QxxV fulcrum is underscored by our finding that N55 forms more hydrogen bonds with the other residues within CDRH2/3 loops (S52, N57, and Q102), while T55 showed fewer interactions with those residues (Figure 7B,C).Collectively, this indicates that the CDRH2 loop with T55 is more flexible to accommodate the changes in Fab binding angles.To determine the structural contribution of N55T for group 1 HA, we resolved a co-complex of H1 trimer (A/Michigan/45/2015) with 09-1B12-UCA + N55T (3.5 Å) and mature 09-1B12 (3.3 Å) (Figure 7D, S1).Notably, group 2 bnAbs can be prevented from recognizing the group 1 stem by clashing with conserved HA1 N-linked glycans 32,34,35 .We find that N55T driven flexibility enables dual recognition of group 1 HA through a mechanism we term 'fulcrum release'.Here the group 1 IAV N-glycans at positions N289, N278, and N33 from the neighboring protomer interact with the antibody LC, tilting the antibody contact angle backward (Figure 7D).Fulcrum release enables UCA + N55T (Figure 7E) and 09-1B12 (Figure 7F) to accommodate this tilt and approach the central stem epitope and to avoid clashing with the group 1 N-glycans.Hence, we provide the structural basis for a single amino acid mutation in the germline encoded CDRH2 loop that enables VH1-18 QxxV antibodies to engage group 1 and group 2 HA stems.
N55T is a generic feature that enables cross-group protection by VH1-18 QxxV bnAb class members.
Given that N55T is present in all VH1-18 QxxV bnAbs, we evaluated its contribution to cross-group protection by two other members of this bnAb class: 21-1A01; and 05-2A09 24 (Figure S6).We passively transferred the mature and inferred UCA forms of these antibodies (± N55T) into C57Bl/6 WT mice and then challenged with H3N2 or H1N1 viruses (Figures S6A,B).Akin to our previous findings with 09-1B12 ± N55T (Figure 6C,D), the UCA forms were comparable to the isotype control, and failed to enable statistically significant protection.However, addition of N55T provided significant cross-group protection for >50% of the mice, approaching the activity of mature forms (Figures S6A,B).Hence, we conclude that N55T, identified by selection within the antibody responses of our humanized mouse system, provides a simple and general molecular 'switch' that enables cross-group protection by VH1-18 QxxV bnAbs.

Discussion
Enhancing coverage of influenza virus diversity traditionally involves inclusion of more or different HA antigens in the seasonal vaccine 49,50 .This principle of additivity has been greatly extended by the recent development of an mRNA vaccine that encodes for HA representatives from all influenza A and B virus categories that elicits broad protection in mice 51 .By contrast, a central challenge for antibody-focusing concepts is to elicit broad coverage through a minimal set of rationally designed antigens 1,2 .Indeed, the compression of multiple protein functionalities within a single molecule is an important theme in the design of therapeutics and scalable biologics 52,53 .Our results highlight a simple molecular 'switch' that can be triggered by a single (and simple) recombinant HA immunogen to deliver exceptionally broad coverage across IAV.
In the absence of human bnAb precursors, HA nanoparticle immunogens (including H3ssF and H7ssF) can elicit heterosubtypic immunity against IAV subtypes, however these responses are not cross-group protective 28,[30][31][32]37 . Undr these conditions, elicitation of cross-group IAV antibodies relies on addition: co-formulation of group 1 + group 2 stem immunogens 36 .By contrast, our results demonstrate that vaccine-elicitation of cross-group protective immunity by a single stem immunogen can be enabled by specific germlineencoded features within the human antibody repertoire.We previously reported a humanrepertoire prerequisite for vaccine-expanding group 1 bnAb classes within mice 28,54,55 and there are analogous repertoire-requirements for expanding these bnAbs within non-human primates 33 .A low level of H3 + /H7 + cross-reactive B cells were initially present in the C57Bl/6 repertoire, however these cells likely lacked sufficient antigen affinity and/or frequencies to enable either vaccine protection against group-unmatched IAV or elicitation of high serum titers of central stem bnAbs that could be detected by cryoEMPEM.While there may be limits on our capacity to detect expansion of these host lineages, our results identify a decisive repertoire effect when VH1-18 QxxV bnAb precursors are present at physiological frequency, namely vaccine elicitation of cross-group protecting IAV bnAbs.
Both precursor frequency and BCR affinity for cognate antigen modulate recruitment to B cell GCs after immunization 43,45,56 .In our system, precursors could be expanded into cross-group protective bnAbs over a logfold range of germline affinities for the group 2 stem (10 −6 -10 −7 M).Vaccine expansion of protective bnAbs across this affinity range was likely supported by fundamental avidity factors, such as stem nanoparticle valency and cell surface arrayed BCR [57][58][59][60][61][62][63][64] , and ultimately by the exceptionally low SHM requirement that we find is needed for full protective activity by VH1-18 QxxV class bnAbs.This minimal SHM greatly contrasts the complex affinity maturation pathways seen for human bnAbs against other hypervariable pathogens such as HIV 65,66 and is exemplified by the N55T molecular switch enabling protection against group 1 and 2 IAV.The correlates of protection we observe are also consistent with VH1-18 QxxV bnAb class: broad neutralizing activity against group 2 viruses with non-neutralizing protection from group 1 IAV 24 .
The N55T substitution is not catalyzed by conventional mutation hotspots 67,68 , but it is nevertheless a marker of the human VH1-18 QxxV bnAb class 20,24 .Enrichment of N55T in the GCs expanded within our humanized mouse system pointed to a previously unrecognized functional importance for this signature that we then verified in other VH1-18 QxxV class bnAbs from humans.Our data indicates that this public amino acid substitution provides a 'fulcrum release' action that pivots the antibody to accommodate a conserved group 1 N-glycan and extend 'hardcoded' germline recognition of group 2-only to group 1 + 2 stems.Achieving this clash relief through a quite minor shift in the organization of the antibody paratope further underscores the low threshold for expanding this bnAb class.
It is uncertain how pre-existing immunity to HA antigens from prior infection and/or vaccination will modulate this simple pathway for bnAb development.Obligate recall of strain specific memory B cells through 'primary addiction' or related feedback effects has the potential to hamper expansion of the pathway [69][70][71][72] .However, the structurally similar group 1 stem nanoparticle, H1ssF, does successfully expand germline-encoded group 1 IAV bnAbs in humans 14,15 , suggesting that absence of an 'immunodistractive' HA head domain may be important for avoiding off-target memory recall when pre-existing immunity is present.
Collectively, our studies demonstrate proof-of-concept for vaccine expansion of unusually broad, cross-group protecting IAV bnAbs using a single recombinant immunogen.This is underscored by a simple molecular signature / switch that can be triggered from the human germline antibody repertoire to enable coverage through minimal immunological complexity.

Limitations of study
There are additional contacts that support cross-group-protection by VH1-18 QxxV class bnAbs 20,24 and we have not assigned their hierarchy in relation to fulcrum release.There are also orthogonal/additional, non-VH1-18 QxxV bnAb classes in humans that enable cross-group protection 20,22,24,25,38 and it is not yet clear if single immunogens will be capable of collectively expanding these pathways and if these bnAbs are also enabled by minimal SHM.We have also used germline inferred BCRs as opposed to bona fide bnAb precursors, although we would note that rationally designed germline stimulating immunogens originally based on inferred UCA have thus far all succeeded in selectively expanding their 'authentic' counterparts in human clinical trials 14,15,31,73,74 .Lastly, and as mentioned earlier, have not accounted for prior exposure to influenza virus or seasonal vaccines where different individual immune-histories may skew 'intended' humoral immunity through 'unintended' memory recall [69][70][71][72] .

LEAD CONTACT AND MATERIALS AVAILABILITY
Lead Contact-Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Daniel Lingwood (dlingwood@mgh.harvard.edu).
Materials Availability-There are no restrictions on the availability on the materials used in this study.S4.

•
The complete code used to compute the frequencies of the VH1-18 QxxV class in human IgM repertoires has been deposited in Zenodo and is publicly available from the date of publication.DOI is listed in the key resources table.
• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request

EXPERIMENTAL MODELS AND PARTICIPANT DETAILS
Generation of 09-1B12-UCA knock-in (KI) mice-09-1B12-UCA KI mice were generated following published protocols 39,40 .In brief, the targeting vector 4E10 78 was modified by the incorporation of human rearranged 09-1B12-UCA KI VDJ (heavy chain construct) or VJ (light chain construct) sequences downstream of the promoter region and by elongation of the 5' and 3' homology regions using the Gibson assembly method (NEB).The targeting vector DNA was confirmed by Sanger sequencing (Eton Bioscience Inc.).
Recombinant HA antigens and B cell probes-Recombinant H3 and H7 trimer ectodomains from A/Perth/16/2009 and A/Shanghai/02/2013, along with ferritin nanoparticle display of their trimeric stem domains (H3ssF and H7ssF) were affinity purified following expression in Expi293 cells according to established methodology 37,55,60,79 .The Expi293 cells were transfected with expression vector using the ExpiFectamine ™ 293 Transfection Kit (Thermofisher), which supplies the transfection reagent and enhancer solution.Five days after transfection, the culture supernatants were harvested, filtered (VacuCap 8/0.2 μm filters, Pall Corporation) and buffer exchanged into PBS using a tangential flow filtration system [Pall Corporation; T-Series Centramate cassettes with Omega PES membrane 10 kDa (Cytiva, OS010T12)].For HA trimers, the buffer exchanged supernatant was equilibrated with Ni Sepharose resin (GE Healthcare), whereas Erythrina cristagalli Gel-ECA-Immobilized Lectin (EY Laboratories) was mixed with the buffer exchanged supernatant containing H3ssF or H7ssF.Ni Sepharose was washed with 20 mM imidazole and the HA trimers subsequently eluted with 500 mM imidazole.For H3ssF and H7ssF, the resin was washed with PBS and the HA nanoparticles were eluted with 0.2 M lactose.All proteins were further purified by size exclusion chromatography (SEC) (AKTA pure protein purification system, Cytiva): HA trimers were resolved on a Superdex increase 200 10/300 column (Cytiva) and the stem nanoparticles were separated using a Superose 6 10/300 column (Cytiva).These same expression and purification procedures were applied to central stem epitope KO versions of the HA trimers and HA nanoparticles (containing N-linked glycan at 45 HA2 ) and also for the recombinant H1 and H5 trimers used in this study (A/Michigan/45/2015, A/California/07/2009, A/Indonesia/05/2005).The HA trimers used in this study also contained the Y98F mutation in the RBS to prevent binding to sialyl oligosaccharide and foldon avi his sequence for trimerization/site specific biotinylation/affinity purification 28,55,60,80,81 .
B cell flow cytometry probes for H3ssF, H3ssF-KO, H7ssF, H7ssF-KO (central stem epitope KO = insertion of N-linked glycan at 45 HA2 ) were generated by fluorescently labelled using amine reactive labeling kits (H3ssF-AF594, H7ssF-AF488, H7ssF-KO -AF647 + H3ssF-KO-AF647), as per our established procedure for ferritin nanoparticle-based B cell probes 28 .In some applications, HA trimers were avi-tagged and biotinylated at this site using the enzyme BirA and then flow probes (H3-APC and H7-AF488) were generated by adding fluorescent SA conjugates in five sequential increments (final molar ratio of HA to streptavidin label was 4:1) so as to saturate the site 60 .
Sorted single-cell suspensions were encapsulated and converted into several DNA libraries following the 10x Next GEM Single cell 5' protocol (10x Genomics).Briefly, single cells were isolated with Gel Bead-In-EMulsions (GEMs) using the Chromium controller provided by 10x Genomics, resulting in uniquely barcoded transcriptome for each individual cell.
After initial cDNA amplification and conversion to dsDNA, individual sequencing libraries were generated for gene expression, VDJ repertoires and hashtag oligos 82 .Library quality was analyzed using a Tapestation 4200 (Agilent).Libraries were pooled at a ratio based on depth requirements established by 10x Genomics and subsequently sequenced using a Nextseq2000 sequencer (llumina).Raw base call files generated by sequencing were demultiplexed, aligned and aggregated using the pipeline offered as part of Cell Ranger (10x Genomics).For VDJ repertoire analysis, immunoglobulin v genes were determined by Cell Ranger.Heavy and Light chains were subsequently paired based on 10x barcodes using a custom R script after doublet determination and removal using hashtag antibody sequences and the Seurat R package (https://satijalab.org/seurat/).Chord plots were produced in R circlize version 0.4.15 (https://cran.r-project.org/web/packages/circlize/index.html)package.
VH1-18-QxxV bnAb precursor frequency in humans-To compare VH1-18 QxxV bnAb precursor frequency within our adoptive transfer model to the natural value in humans, we measured this value in publicly available IgM BCR repertoires from n=10 human subjects sequenced to high depth 41 .From this dataset, we considered only those antibody sequences annotated as encoding productive IgM heavy chains.The complete code used to compute the frequencies of the VH1-18 QxxV class in these repertoires is available at https://www.doi.org/10.5281/zenodo.10800716.The Bash, R, and Python languages were used, as well as tidyverse 83 and pandas software packages 84 .

Sequential (2x) immunization of humanized mice and viral challenge-One
day after adoptive transfer of CD45.2 B cells from 09-1B12-UCA KI mice, the recipient CD45.1 mice were given an intraperitoneal injection of 50 μg of H3ssF within 100 ml of inoculum containing 50% w/v Sigma adjuvant (Sigma, Cat# S6322; also known as Ribi), or Sigma adjuvant-only.We also included a CD45.1 mouse group that received H3ssF, but no UCA cells were present.Mice were boosted 42 days after the initial immunization (H3ssF + Sigma adjuvant for the mice primed with H3ssF; or Sigma adjuvant-only for the non-immunogen group).Fourteen days after the boost (day 56), the mice were intranasally infected with 100% lethal doses of either: subtype-matched H3N2 X-31 (BEI Resources cat# NR-3483) (10 8 TCID 50 /ml); or group-unmatched, mouse-adapted H1N1 A/ California/07/2009 (maA/Cal/09) 75,76 (10 4 TCID 50 /ml).Mice were monitored each day for loss.Both viruses were cultured in MDCK cells and quantified by TCID 50 in MDCK cells 85 .X-31 was obtained from BEI Resources and maA/Cal/09 was kindly provided by Sabra Klein and Andrew Pekosz, John Hopkins University.
BCR sequencing of GC B cells-Single cell BCR libraries were generated from products of whole transcriptome amplification (WTA) using the Smart-Seq2 protocol 86 .
The WTA product from each single cell reaction were first subjected to two 0.8x (v/v) SPRI bead-based cleanups followed by cDNA quantification/normalization.BCR sequences from heavy and light chains were enriched from single B cells using a V region and J region specific primer set in which the primers were also attached to Illumina P7 (V region) and P5 sequences (J region) (final concentration: 0.5 μM each) (Table S5).After amplification, a 0.8x (v/v) SPRI cleanup was performed, and we quantified and normalized the amplicons to 0.2-0.5 ng/μL.Within a subsequent step-out PCR [Kapa HiFi HotStart ReadyMix; Kapa Biosystems], we added cellular barcodes and Illumina sequencing adapters (based on Nextera XT Index Adapters, Illumina Inc.) to each single cell-amplified heavy and light chain, as we have performed previously 28,55,61 .After a 0.8x (v/v) SPRI cleanup, the heavy chain and light chain products were pooled and sequenced using paired end 250×250 reads and 8×8 index reads on an Illumina MiSeq System [MiSeq Reagent Kit v2 (500-cycle)].
The BCR heavy and light chains were paired and reconstructed using PandaSeq 87 and aligned against the human IMGT database 88,89 with PCR/sequencing error correction using MigMAP (a wrapper for IgBlast: https://github.com/mikessh/migmap). Consensus V-chain and L/k-chain sequences for each single cell was performed by collapsing all reads with the same CDR3 sequence and then identifying the top heavy and light chain sequences based on frequency.Any heavy or light chain sequence with fewer than 15 reads or a frequency less than twice that of the next sequence of the same chain was considered without consensus.Phylogenetic trees to visualize relatedness were generated from the heavy chain nucleotide sequences using the maximum likelihood method with the Tamura-Nei model in MEGA11 software 90,91 .Heavy chain and light chain sequences from 09-1B12-UCA were used as the baseline for tree construction.Sequence logos were generated using WebLogo (https:// weblogo.berkeley.edu) 92.
Influenza reporter viruses were generated as described previously 46 .Briefly, H1N1 and H3N2 viruses were generated using plasmids encoding bidirectional cassettes for PB2, PA, NP, M, and NS from A/WSN/1933 96 , tdKatushka2 containing PB1 packing sequences, a plasmid encoding human TMPRSS2, and HA and NA segments flanked with non-coding regions from A/WSN/1933 (for H1N1 viruses) or A/Netherlands/009/2010 (for H3N2 viruses).Plasmids were transfected into HEK293T-PB1 cells in a 6-well plate using TransIT-LT1 (MirusBio, #2306).After three days, the supernatant was clarified by centrifugation at 800 ×g for 5 minutes and added to a monolayer of MDCK-SIAT1-PB1 cells in a 6-well plate.Two to three days later when cytopathic effect was evident, the supernatant was clarified and added to a monolayer of MDCK-SIAT1-PB1 cells in T75 or T150 flasks.Two to three days later, the supernatant was clarified, and aliquots of viral stocks were frozen at −80 °C.To generate H5N1 and H7N9 viruses, the internal genes (PB1, PB2, PA, NP, M, NS) from A/Puerto Rico/8/1934 were used with tdKatushka2 bearing the HA packing sequences from A/Puerto Rico/8/1934.For H5N1 and H7N9 viruses, a plasmid expressing H5 HA (A/Vietnam/1203/2004) or H7 HA (A/Shanghai/02/2013) were additionally transfected into HEK293T cells.H5N1 and H7N9 viruses were passaged and propagated as above in MDCK-SIAT1-HA cells constitutively expressing H5 or H7 HA.
One day prior to titering, MDCK-SIAT-PB1 (or -HA) cells were seeded in 96-well plates at 20,000-30,000 cells per well (CellVis, #P96-1.5P).The next day, viral stocks were thawed and diluted 2-fold 23 times in quadruplicate.The virus dilutions were then mixed with an equal volume of flu media additionally supplemented with 2 μg/ml of TPCK-treated trypsin (Sigma, #T1426).Virus was incubated at 37 °C / 5% CO 2 for 1 hour before removing flu media from MDCK-SIAT1-PB1 (or -HA) cells and adding 100 μl of the virus.Approximately 18-20 hours later, the cells in each well were imaged on a CellDiscoverer7 (Zeiss) instrument and fluorescent cells were counted.The cell counts were plotted, and a sigmoidal curve was fitted in GraphPad Prism.The dilution that corresponded to the half maximal positive cell count was used for subsequent neutralization assays.
For microneutralization measurements, the viruses were diluted in flu media with 2 μg/ml of TPCK-treated trypsin and antibodies were diluted in a four-fold series in flu media.The virus and antibody dilutions were mixed in equal volumes in a 96-well plate and incubated at 37 °C / 5% CO 2 for 1 hour.The flu media was then removed from the MDCK-SIAT1-PB1 (or -HA) cells and the antibody/virus mixture was added.Approximately 18-20 hours later, the cells were counted as above.Each plate contained ten virus only control wells and two cell only controls.Each value had the cell only control background subtracted and was normalized to the average of the virus only controls.The percent neutralization was plotted and fitted with a sigmoidal curve (GraphPad Prism) to determine the half maximal inhibitory concentration (IC 50 ).Each neutralization assay was run in duplicate.

Immunization of humanized mice and serum processing for CryoEMPEM-One
day after adoptive transfer of CD45.2 B cells from 09-1B12-UCA KI mice, the recipient CD45.1 mice were immunized intraperitoneally with either 50 μg H3ssF or 50 μg H7ssF; each delivered in a 100 ml inoculum containing 50% w/v Sigma adjuvant.The mice were boosted again at 42 days with these same inoculums and immune sera were obtained at 15 and 28 days post-boost.These sera were then processed for cryoEMPEM analysis 48 .Briefly, Serum IgG was purified from pooled sera (in the same group) with Protein G resin and subsequently digested with papain.Digested polyclonal Fabs were purified over Superdex 200 Increase column (Cytiva).
CryoEM-For cryoEM, HA (30 μg) was incubated with the purified monoclonal Fab (40 ug) or polyclonal Fabs (0.5-1 mg) overnight at 4 °C.The complex was then purified over Superdex 200 Increase column (GE Healthcare) and concentrated to ~0.7 mg/ml.Next, 3 μL of the complex was mixed with 0.5 μL 0.7% (w/v) Octyl-beta-Glucoside (OBG) before deposition onto glow-discharged 1.2/1.3Cu 300 grids (EMS), directly preceding the deposition in a Vitrobot (Thermo Fisher Scientific) with following settings: 4°C, 100% humidity, 0 s wait time, 4.5-6 s blot time, blot force 1.Once sample was deposited, the grids were blotted and plunged into liquid to immobilize the particles in vitreous ice.The micrographs were collected with EPU image acquisition software at a nominal magnification of ×190,000 with a TFS Falcon 4 detector mounted on a Glacios set to 200 kV set to counting mode, with a total exposure dose of ~50 e − /Å 2 .Micrographs were collected in CryoSPARC Live and followed by gain reference correction, motion correction, defocus estimation, and micrograph curation (Table S1).Template Picker was used to pick particles, which were then extracted and 2D-classified in cryoSPARC.The particles in selected 2D classes were further cleaned up by Heterogenous refinement using C1 symmetry and 3D Variability with symmetry expanded particles.Final maps were made by homogeneous refinements with tight masks on HA and Fab on one protomer.
X-ray diffraction data was collected at beam line 24-ID-E (Advanced Photon Source) and processed with XDSGUI (https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDSGUI).The work used NE-CAT beamlines (GM124165), a Pilatus detector (RR029205), and an Eiger detector (OD021527) at the APS (DE-AC02-06CH11357).The structure was initially solved by molecular replacement using the HA protomer from PDB 4KVN, 16.a.26variable heavy chain with the CDRH3 removed (from PDB 5K9Q), and a model of the variable light chain with the CDRL3 removed 101 .PHENIX 102 was used to initially refine the coordinates and B factors prior to manual model building in COOT 103 .Final refinement additionally included Translation Libration Screw refinements and Ramachandran restraints (Table S2).The final model was deposited to the Protein Data Bank (8UWA).
Structure Modelling and MD simulations-To characterize the interaction profiles of the UCA/09-1B12 antibodies in complex with H1, H3 and H7 we performed molecular dynamics simulations of these antibody complexes, respectively.As starting structures for our simulations, we used the available cryo-EM structures, presented in this study.For all these antibody-antigen complexes we performed each three replicas of 1μs classical molecular dynamics simulations.Furthermore, we also performed simulations of the free variable domains (Fvs), namely 09-1B12-UCA, 09-1B12-UCA + N55T and 09-1B12, to characterize the effect of the mutations on the dynamic properties of the Fvs.
The starting structures for our simulations were prepared in MOE using the Protonate3D tool 104,105 .With the help tool of the Amber Tools20 package 106 , we explicitly bonded all existing disulfide bridges and placed the antibody-antigen complexes into cubic water boxes of TIP3P water molecules with a minimum wall distance to the protein of 12 Å 107 .Parameters for all antibody-antigen and free Fv simulations were derived from the CHARMM36m 108 .To neutralize the charges, we used uniform background charges 106,109 .
Each system was equilibrated using a multistep equilibration protocol 110 .Molecular dynamics simulations were performed using pmemd.cuda in an NpT ensemble to be as close to the experimental conditions as possible and to obtain the correct density distributions of both protein and water 111 .Bonds involving hydrogen atoms were restrained by applying the SHAKE algorithm 112 , allowing a timestep of 2.0 fs.Atmospheric pressure of the system was preserved by weak coupling to an external bath using the Berendsen algorithm 113 .The Langevin thermostat was used to maintain the temperature at 300K during simulations 114,115 .
To calculate contacts of the antibody-antigen complexes, we used the GetContacts software (https://getcontacts.github.io/).This tool can compute interactions within one protein structure, but also between different protein interfaces and allows to monitor the evolution of contacts during the simulation.The contacts are defined based on the default geometrical criteria provided by GetContacts.
The reaction was in PBS containing 1 mM EDTA, with a ratio of 5 mg of Lys-C per milligram of IgG.After incubating for 12 hours at room temperature, the reaction was stopped by addition of 1x complete protease inhibitor cocktail (Roche, Cat # 11697498001) and Protein A/G-agarose was applied to remove any uncleaved IgG.The resin was washed with PBS, and the supernatant was concentrated using Amicon Ultra concentrators with a 10 kDa cutoff.The Fabs were further purified by SEC, using a Superdex 200 10/300 column (GE Healthcare).BLI was performed using the Personal Assay BLItz System (Fortebio).Following avi-tag biotinylation (see earlier), the biotinylated forms of HA trimers spanning group 1 and group 2 diversity (A/Perth/16/2009, A/Shanghai/02/2013, A/Michigan/45/2015, A/California/07/2009, A/Indonesia/05/2005) were immobilized onto streptavidin biosensors (Sartorius, Cat#18-5019).After establishing a baseline in kinetic buffer (PBS containing 0.02% Tween20, 0.1% BSA), the Fabs were introduced at 0.625 mM, 1.25 mM, 2.5 mM, and 5 mM, with 120 seconds of association and 120 seconds of dissociation.The equilibrium dissociation constant (KD) values were calculated by fitting a 1:1 binding isotherm using software provided by the vendor 77 .

QUANTIFICATION AND STATISTICAL ANALYSES
All statistical analysis were conducted using Prism 9.01 (GraphPad).Sample sizes and statistical tests are indicated in the figure legends.Data were considered statistically significant at P<0.05.

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Natural HA affinity for human bnAb precursors is transduced into vaccine nanoparticles

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A humanized mouse is engineered to contain human bnAb precursor frequency

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Vaccine protection against Group 1 + Group 2 IAV is generated by stem bnAb elicitation

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The antibodies undergo a single amino acid 'switch' to accommodate IAV glycan diversity  S3).See also        Arrows indicate the flexible antibody tilting enabled by fulcrum release to accommodate the conserved group 1 IAV glycan positions.See also Figure S1, Tables S1, S2.
Figure S1 and Tables S1, S2 (in relation to B,C).

Figure 3 .
Figure 3. Stem nanoparticles selectively expand VH1-18 QxxV bnAb precursors from physiological frequency in the antibody repertoire and induce diversification through somatic hypermutation in GCs.(A) Schematic of adoptive transfer performed at precursor frequencies of ~1 per 10 5 09-1B12-UCA B cells into WT mice at day −1 and subsequent single immunization of higher affinity H3ssF or lower affinity H7ssF.Naked ferritin particles were also given as a control.All vaccines were adjuvanted by the Sigma Adjuvant System.Spleens were sampled at the time points indicated.(B) Representative flow plots of CD45.2 B cells being recruited to GCs at days 8, 15, and 28 post-vaccination.(C) The percentage of GC B cells in the CD45.1 host was quantified at each time point (n=5 mice per immunogen, mean ± SD, one experiment).(D) The percentage of CD45.2 B cells within the host GCs each time point (n=5 mice per immunogen, mean ± SD, one experiment).(E) The GC CD45.2 B cells were also marked by epitope specificity to the central stem site [H3ssF + /H7ssF + /H3ssF-KO − / H7ssF-KO − (central stem epitope KO =N-linked glycan at 45 HA2 )] and single GC CD45.2 B cells in this gate were sorted by FACS and subjected to BCR sequencing.Results presented in B-E were recapitulated if H7 and H3 trimers were used (instead of nanoparticles) as the antigen B cell probes (Figure S5C-G).(F, G) HC nucleotide diversification of H3ssF + /H7ssF + /H3ssF-KO − /H7ssF-KO − B cell clones at 28 days after immunization with H3ssF or H7ssF.(H) Mutation frequency in the HC amino acid sequence at 28 days post

Figure 5 .
Figure 5. Stem focused VH1-18 QxxV responses enrich for the public mutation N55T within germinal centers and serum antibodies elicited by stem nanoparticles.(A, B) Logo plots of the CDRH2 domain of the VH1-18 QxxV class precursors enriched in the GCs at 15 and 28 days post-immunizations with H3ssF or H7ssF (see also Figure 3F-K; 167 GC BCRs for H3ssF at Day 15 (n=3 mice), 125 GC BCRs for H3ssF at Day 28 (n=3 mice), 99 GC BCRs for H7ssF at Day 15, 111 GC BCRs for H7ssF at Day 28 (n=3 mice); one experiment).(C-I) CryoEMPEM was performed on VH1-18 QxxV antibodies elicited in the serum after sequential immunization (day 0 prime + day 42 boost) with either H3ssF or H7ssF.In all cases immune sera were evaluated at 15 and 28 days post-boost.(C) H3ssF elicited IgG showing differential reactivity to H3 ± epitope KO (central stem epitope KO= N-linked glycan at 45 HA2 ); adjuvant only is the control (n=9 at Day 15 and n=9 mice at Day 28; n=6 control mice received adjuvant only; one experiment).(D) CryoEMPEM of H3ssF immune sera at 15 and 28 days post-boost, with antibodies in complex with H3 trimer (A/Perth/16/2009) [immune sera pooled from all mice at Day 15 (one experiment); immune sera pooled from all mice at Day 28 (one experiment)].The 09-1B12-UCA Fab was docked into the maps and the HC N55 residues are boxed in red.(E) No density of 09-1B12-like Fab in complex with H3 trimer was found in the immune sera from WT C57Bl/6 mice (lacking VH1-18 QxxV bnAb precursors) subjected to the same H3ssF immunization regimen (28 days post-boost is shown) (immune sera pooled from n= 10 mice,
2 +/+ ) colony were bred at the animal facility of the Gene Modification Facility (Harvard University) and breeding for colony expansion and experimental procedures was subsequently performed at the Ragon Institute of Mass General, MIT, and Harvard.Ear or tail snips from 09-1B12UCA KI mice were genotyped by TaqMan assay under a fee for service agreement (TransnetYX).TaqMan probes for the genotyping assay were developed by TransnetYX.All experiments were performed under the approval by the Institutional Animal Care and Use Committee (IACUC) of Harvard University and the Massachusetts General Hospital (MGH) (Animal Study Protocols 2016N000022 and 2016N000286, 2014N000252) and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).Both male and female animals were used at 8-12 weeks of age.The light cycles in the animal room were set on a 12 hour light cycle [7AM-7PM (ON) 7PM-7AM (OFF)].The temperature range for the room was 68 -73 degrees Fahrenheit and the humidity index was from 30% -70%.The feed was replaced every two weeks with fresh pelleted ration (Prolab Isopro RMH 3000), concomitant with changing fresh bedding in the cage.The cages were also inspected daily and additional pellets were added if food was low or empty.METHOD DETAILSAdoptive transfer-For experiments male B6.SJL-Ptprc a pepc b /BoyJ mice (CD45.1 +/+ ) 8-12 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME).CD45.2 + B cells from male or female 09-1B12-UCA KI mice (H 09-1B12-UCA/WT , κ 09−1B12- UCA/WT ) were enriched using the Pan B Cell Isolation Kit II (Miltenyi Biotec), counted, diluted to desired cell numbers in PBS and adoptively transferred into CD45.1 + recipient mice as reported previously + / H3ssF-KO − /H7ssF-KO − ) in the spleen of the recipient mice.Transfer of 100,000 09-1B12-UCA B cells reproducibly gave a value of ~1 in 100,000 B cells (n=5 recipient mice per transfer amount, one experiment), consequently this was the VH1-18 QxxV bnAb precursor frequency used in all subsequent immunization experiments.(H) 09-1B12-UCA B cells were transferred (or not transferred) Phosphatase AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific Immunity.Author manuscript; available in PMC 2024May 16.